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Genes
Introduction CDH has been observed in many single gene disorders suggesting a role for these genes in diaphragm development. Numerous CDH Animal Models also point to interesting candidate genes potentially involved in the pathogenesis of CDH. Slavotinek has reviewed single gene disorders associated with CDH Bielinska et al. have reviewed the molecular genetics of CDH Single Gene Disorders Candidate Genes for Isolated CDH Mutation Analysis of (Isolated) CDH Patients Mutation analysis studies of candidate genes for isolated CDH patients are limited. We will focus here on novel genes which are potential candidates for causing isolated CDH, rather than the syndromes with which CDH has been associated. Identifying genes directly involved in diaphragm development may allow for the identification of potential therapeutic targets for particular patients. The findings from these studies are listed below; Candidate CDH gene FOG2 FOG2; cytosine to thymine heterozygous change in exon 4 predicting R112X The FOG2 gene was sequenced in 30 (deceased) CDH patients; Of these 30 cases, 17 (57%) had Bochdalek CDH, 2 (7%) had agenesis of the diaphragm, 7 (23%) had eventration of the diaphragm or muscularization defects (without Bochdalek CDH), and 4 (13%) had Bochdalek hernia of one hemidiaphragm and an eventration of the other. Sequencing of FOG2 revealed a de novo heterozygous nonsense mutation in a patient who died at birth with severe pulmonary hypoplasia and a posterior diaphragmatic eventration. This sequence change was not present in the analysis of DNA from 400 normal adults. Candidate CDH genes FOG2 & PDGFRalpha FOG2 '; c.A2107C predicting p.M703L; c.A2527G predicting p.T843A '''PDGFRalpha '; c.C2889G predicting p.L967V All coding exons and exon–intron boundaries of both FOG2 and PDGFRa were sequenced in 96 patients (except exon1 of FOG2, for which sequence data was generated for 56 patients due to high GC content of this region). Of 96 patients, 65 (68%) had left-sided CDH, 22 (23%) had right-sided CDH, one case had absence of the posterior lip of the diaphragm, one patient had a retrosternal hernia, and one had an eventration (location not specified) and an antral diaphragmatic hernia (1% each). The type of diaphragmatic defect was unknown in six patients (6%). Fifteen hernias were described as Bochdalek CDH (16%) and included diaphragmatic defects that involved either the left or the right side. Mutations of FOG2 were identified in 2 patients, 1 left-sided CDH & 1 right-sided CDH. The patient with a PDGFRalpha mutation had left-sided CDH with multiple additional anomalies comprising pulmonary hypoplasia, congenital heart disease with a ventricular septal defect, secundum type atrial septal defect and tricuspid regurgitation, malrotation of the intestines, a multicystic liver with an abnormal biliary tree, a short neck with slight webbing, a small chest cavity with abnormal thoracic vertebrae, including butterfly vertebrae, and multiple rib anomalies, dysmorphism with a prominent, sloping forehead, small and downslanting palpebral fissures with sparse eyebrows, a flat and broad nasal root, low-set ears that had thick, protruding helices, bilateral simian creases, fifth finger clinodactyly, adducted thumbs and streaky hyper and hypopigmentation of the skin. A skin karyotype showed 46,XX with 1/50 cells showing mosaicism for trisomy 15 For p.M703L in FOG2, a total of 526 control chromosomes were screened. For p.T834A in FOG2, a total of 564 control chromosomes were screened. For p.L967V in PDGFRalpha'','' a total of 768 control chromosomes were screened. Family members were unable to be contacted to verify if alterations were de novo Candidate genes in the 15q26 genomic region '''SIAT8B CHD2 MCTP2 NR2F2 ARRDC4 RGMA Mutation analysis was performed in at least 100 CDH patients per gene. Array CGH was performed for 29 CDH patients, 16 non-isolated CDH & 13 isolated CDH. Six of these patients had previously detected cytogenetic aberrations. None of these alterations were present in more than 100 Mexican American control chromosomes. The SIAT8B ''gene has since been excluded from the 15q26 CDH-critical region by further mapping studies. The authors were unable to conclude that the alterations in any of the sequenced genes are pathogenic. Candidate genes in the 1q41-q42 region '''DISP1 '; c.4412C>G predicting p.Ala1471Gly. HLX 179 CDH patients were studied, ~2/3 were classed as isolated CDH, ~1/3 complex CDH. All patients were initially screened for copy number changes of the 1q41-1q42.12 genomic region by MLPA. 2 patients displayed microdeletions of this region. In one patients, a de novo heterozygous C–G transversion (c.4412C>G), was identified; this mutation was predicted to change an alanine to glycine at position 1471 (p.Ala1471Gly). The prenatal phenotype was left-sided Bochdalek CDH, fetal heart abnormalities (ventricular septal defect and abnormal aorta), and a left-sided cleft lip with bilateral cleft palate. This mutation was also observed to be present at different levels of mosaicism in different tissues analysed. The authors conclude that this represents the first de novo DISP1 point mutation in a patient with complex CDH. Combining this finding with Disp1 embryonic mouse diaphragm and lung tissue expression, as well as previously reported human chromosome 1q41q42 aberrations in patients with CDH, suggests that DISP1 may warrant further consideration as a CDH candidate gene Two additional DISP1 variants, p.Met1096Thr and p.Asn1281Ile, were identified in three and two patients, respectively. These variants are predicted to be “benign”or “tolerated”, and were inherited from a healthy parent in three families with available parental samples. In a patient with isolated CDH, a previously unreported single nucleotide variation (c.27C>G) resulting in a change from phenylalanine to leucine at amino acid position 9 (p.Phe9Leu) was identified in the HLX gene. However, this was shown to be inherited from a phenotypically normal mother. 96 control chromosomes were screened. Candidate gene FGFRL1 in the 4p16.3 genomic region FGFRL1 The candidate gene FGFRL1 within the 4p16.3 genomic region was sequenced in 54 patients with isolated CDH. 6 known coding SNPs were identified: c.209G > A (p.Pro20Pro), c.977G > A (p.Pro276Pro), c.1040T > C (p.Asp297Asp), c.1234C > A (p.Pro362Gln), c.1420G > T (p.Arg424Leu), and c.1540C > T (p.Pro464Leu). No novel SNPs or gene mutations were identified. Additional non-isolated CDH patients were then genotyped for four of these six SNPs, including the three non-synonymous SNPs, to make a total of 200 chromosomes. The allele frequency for the four SNPs, did not differ significantly between patients and normal controls (p'' ≥0.05). 109 control samples were analysed. The authors also applied cDNA expression arrays to diaphragms from Fgfrl1 homozygous null mice. This showed a significant decrease in the expression level of the ''Lrtm1 gene in E18.5 when compared with wildtype littermates. Lrtm1 shows similarity with Slit3, which is involved in cell motility and has previously been implicated in central tendon hernias in the murine diaphragm, and can alter the expression of two other genes, Rac and Cdc42, that are essential for myoblast fusion. The authors conclude that it remains to be seen whether Lrtm1 is of similar function to Slit3, and whether Fgfrl1 can contribute to diaphragm hypoplasia through a mechanism involving cell motility and/or myoblast fusion. References